Duchenne muscular dystrophy (DMD) is a muscle wasting disease caused by mutations in the DMD gene, which encodes dystrophin. There are presently no cures. The current standard of care is corticosteroid treatment, which delays the progression of skeletal muscle and cardiac dysfunction but also has serious side effects. Through the use of animal DMD models, in particular the mdx mouse, the GRMD dog, and the zebrafish dmd mutant, many promising alternative pharmacological therapies have already been identified. Many of these drugs benefit DMD by modulating pathological mechanisms downstream of the DMD gene. The current challenge is to determine how to best translate preclinical pharmacological studies to DMD patients. One critical issue is that, because many DMD patients take corticosteroids as well as cardiac medication, any new pharmacological therapies should be compared to, and tested for efficacy in the presence of, these current treatments. A second issue is that the potential for novel drug combination therapies for DMD has not been fully explored. Drug comparison and combination studies are possible in the mdx mouse, but face many challenges. To increase the probability of success for translation of pharmacological therapies, preclinical studies should be validated in multiple animal models. The long-term goal of this proposal is to establish zebrafish as a preclinical translation model fo evaluating optimal DMD drug combination therapies. The hypothesis of this proposal is that the zebrafish dmd model can be used to identify both beneficial drug combinations that ameliorate DMD and biomarkers associated with this amelioration. Zebrafish offer several advantages for this study, in particular that both cardiac and skeletal muscle phenotypes and biomarkers can be easily monitored. This proposal will address two Specific Aims. Aim 1 will systematically evaluate a set of drug combination therapies using the zebrafish dmd model. The drugs to be tested are already in use on DMD patients or are in clinical trials. Multiple outcomes will be examined: muscle structure, muscle function, cardiac structure, and survival. Aim 2 will test for mRNA biomarkers associated with disease progression and drug- induced disease amelioration using qRT-PCR and RNA-seq on dmd zebrafish. One expected impact of this project will be to show that dmd zebrafish can replicate, and therefore potentially be predictive of, mammalian DMD drug combination effects and biomarkers. A second potential impact will be the identification of novel drug combinations that are beneficial for DMD. Because this study will test drugs that are already being used in the clinic or in clinical trials, the findings could potentialy be rapidly incorporated into drug-combination therapies for patients. A third impact is that the analysis of the dmd cardiac phenotype and cardiac mRNA markers will address a critical knowledge gap in DMD cardiac issues. The long-term impact of this project will be to advance a critical DMD animal model for comparative drug validation.